There are literally hundreds of millions of tests of aqueous fluids conducted each year. This is particularly true in the medical field where urine, blood, serum, and other bodily fluids are tested for literally hundreds of different substances.
Many of these substances can be detected using particular electrodes with membrane bound enzymes which react with a substance to be detected and produce a detectable product such as hydrogen peroxide or oxygen. Such electrode systems are reliable but very limited in application. Since a membrane bound enzyme is required, the electrode is dedicated for analysis for one particular substance. This in turn causes the testing to be relatively expensive.
There are a number of different enzymatic assays and immunoassay that are employed which produce a nicotinamide adenine dinucleotide such as nicotinamide adenine dinucleotide (NADH) or nicotinamide adenine dinucleotide phosphate (NADPH) as products in quantitative amounts which can then be detected. These detection systems currently rely on photometric detection of NADH and NADPH.
Photometric detection of NADH and NADPH is problematic. Depending on the fluid being tested, various pretreatment steps are required in order to permit the NADH or NADPH to be detected photometrically. For example, if the sample is blood, red blood cells will interefere with colorimetric detection systems. Therefore, the blood cells must be removed. If a sample incorporates excessive amounts of protein, lipid, bilirubin or hemoglobin, these can interfere with certain photometric detection systems. Thus, depending on the particular test required, the sample will require various pretreatments. This requires unique treatment of each sample for each test method. This significantly increases the difficulty and the expense of sampling.
Particularly in hospitals this presents additional problems. Handling of any bodily fluid is potentially hazardous. Worker safety is extremely important and any time a bodily fluid such as blood is handled, there is a potential that the worker will be infected with a virus, such as the AIDS virus or hepatitis. Therefore, it is desirable to minimize any handling of these hazardous test samples.
NADH and NADPH are theoretically detectable using electrochemical analysis. Direct oxidation of NADH or NADPH is an attractive method, but it has general limitations. First, the high applied potential necessary for the direct oxidation of NADH and NADPH due to the large over-voltage at solid electrodes compromises selectivity due to interfering oxidation reactions from serum components, such as uric acid, ascorbic acid, and acetaminophen. Second, the detection limit of an electroanalytical technique is generally not as good at such a positive potential due to high background currents. Further, proteins associated with certain samples can coat an electrode surface and thereby inhibit the detection of NADH or NADPH.
The large positive potential required to oxidize NADH at a solid electrode is due to the overpotential associated with a slow heterogeneous electron transfer. This overpotential has been diminished by chemical modification of the electrode with appropriate catalysts such as orthoquinones covalently bound to the carbon electrode, phenazonium salt adsorbed on graphite, dopamine covalently attached to vitreous carbon, catechols with pyrene side chain adsorbed on graphite and amide linked 3,4 dihydroxy benzylamine and vinyl polymerized eugenol immobilized physically on carbon. Nile blue has been coupled with terephthaloyl chloride to form a compound that adsorbs to graphite electrodes and shifts the NADH oxidation wave to -0.15 volts. However, the catalytic action of these electrodes has generally proven to be too short lived for their practical use in a flow system or repetitive analysis of many samples containing NADH. Accordingly, electrochemical analysis of NADH and NADPH has proved unsatisfactory and therefore photometric methods have been required with all their attendant disadvantages.